Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology

At present, only few methods for the effective assembly of multigene constructs have been described. Here we present an improved version of the MultiRound Gateway technology, which facilitates plant multigene transformation. The system consists of two attL-flanked entry vectors, which contain an attR cassette, and a transformation-competent artificial chromosome based destination vector. By alternate use of the two entry vectors, multiple transgenes can be delivered sequentially into the Gateway-compatible destination vector. Multigene constructs that carried up to seven transgenes corresponding to more than 26 kb were assembled by seven rounds of LR recombination. The constructs were successfully transformed into tobacco plants and were stably inherited for at least two generations. Thus, our system represents a powerful, highly efficient tool for multigene plant transformation and may facilitate genetic engineering of agronomic traits or the assembly of genetic pathways for the production of biofuels, industrial or pharmaceutical compounds in plants.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Using a modified TA cloning method to create entry clones

We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on alpha-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility.     

利用TA克隆的方法简便构建入门克隆

Gateway技术是一种通用型克隆方法,其基于λ噬菌体位点特异性重组,将目的DNA快速克隆到各种与Gateway技术兼容的目的载体上,不需要进行酶切和连接反应。但存在获得入门克隆过程中相关反应酶制剂价格昂贵,且药品订购时间较长等问题。通过对入门载体pDONR207的改造,使之产生3’端具有单个T 末端的线性化的入门载体,采用TA克隆的方法替代BP反应,从而简便、经济和快速地获得入门克隆。利用改造后的Gateway技术构建拟南芥SOS2基因的原核表达载体和真核表达载体,通过原核表达和原生质体瞬时表达证明通过此方法构建的表达载体在原核细胞和真核细胞中都得到了很好的表达。     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning

Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.     

Enabling technologies for manipulating multiple genes on complex pathways

Many complex biochemical pathways in plants have now been manipulated genetically, usually by suppression or over-expression of single genes. Further exploitation of the potential for plant genetic manipulation, both as a research tool and as a vehicle for plant biotechnology, will require the co-ordinate manipulation of multiple genes on a pathway. This goal is currently very difficult to achieve. A number of approaches have been taken to combine or `pyramid' transgenes in one plant and have met with varying degrees of success. These approaches include sexual crossing, re-transformation, co-transformation and the use of linked transgenes. Novel, alternative `enabling' technologies are also being developed that aim to use single transgenes to manipulate the expression of multiple genes. A chimeric transgene with linked partial gene sequences placed under the control of a single promoter can be used to co-ordinately suppress numerous plant endogenous genes. Constructs modelled on viral polyproteins can be used to simultaneously introduce multiple protein-coding genes into plant cells. In the course of our work on the lignin biosynthetic pathway, we have tested both conventional and novel methods for achieving co-ordinate suppression or over-expression of up to three plant lignin genes. In this article we review the literature concerning the manipulation of multiple genes in plants. We also report on our own experiences and results using different methods to perform directed manipulation of lignin biosynthesis in tobacco.     

单子叶植物RNA干扰和过表达Gateway载体的构建

基因枪和农杆菌介导的遗传转化是目前常用的两种单子叶植物遗传转化方法。载体的发展和改良是提高植物遗传转化效率的重要基础,RNA干扰载体和过表达载体是目前通过遗传转化研究植物基因功能的主要工具。Gateway克隆技术是一种基于lambda噬菌体特异位点重组特性的通用克隆技术,该技术可以将大批目的基因方便、快捷地连接到受体载体上。本文利用Gateway技术结合传统酶切、连接方法,构建了适用于单子叶植物基因枪和农杆菌转化的RNA干扰Gateway载体pAHC-PSK-RNAi、pClean-G185-RNAi和过表达Gateway载体pAHC-PSK-OE和pClean-G185-OE,为利用基因枪和农杆菌介导的遗传转化,在小麦和水稻等单子叶植物中进行规模化基因功能研究奠定了基础。     

Construction of two Gateway vectors for gene expression in fungi

We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.